LETTERS https://doi.org/10.1038/s41588-020-0643-0 CTCF is dispensable for immune cell transdifferentiation but facilitates an acute inflammatory response Grégoire Stik 1 ✉ , Enrique Vidal 1, Mercedes Barrero1, Sergi Cuartero1,2, Maria Vila-Casadesús1, Julen Mendieta-Esteban 3, Tian V. Tian1,4, Jinmi Choi5, Clara Berenguer1,2, Amaya Abad1, Beatrice Borsari 1, François le Dily 1, Patrick Cramer 5, Marc A. Marti-Renom 1,3,6, Ralph Stadhouders 7,8 ✉ and Thomas Graf 1,9 ✉ Three-dimensional organization of the genome is important transcriptional rewiring during cell-state transitions—often accom- for transcriptional regulation1–7. In mammals, CTCF and the panied by extensive cell division—remains controversial24. cohesin complex create submegabase structures with ele- We have recently developed a system that is particularly suit- vated internal chromatin contact frequencies, called topo- able for studying the role of CTCF in cell-state transitions, which logically associating domains (TADs)8–12. Although TADs consists of a B leukemia cell line (BLaER) that can be efficiently can contribute to transcriptional regulation, ablation of converted by exogenous CEBPA expression into functional induced TAD organization by disrupting CTCF or the cohesin com- macrophages (iMacs) with only one cell division on average (Fig. 1a plex causes modest gene expression changes13–16. In con- and Supplementary Note 1)25. Using this system, we analyzed a trast, CTCF is required for cell cycle regulation17, embryonic time series of transdifferentiating cells for genome-wide changes development and formation of various adult cell types18. To in three-dimensional (3D) genome organization (in situ Hi-C), uncouple the role of CTCF in cell-state transitions and cell pro- enhancer activity (ChIP–seq of histone modifications), chromatin liferation, we studied the effect of CTCF depletion during the accessibility (ATAC-seq) and gene expression (RNA-seq). conversion of human leukemic B cells into macrophages with We first determined genome segmentation into A and B com- minimal cell division. CTCF depletion disrupts TAD orga- partments on the basis of the first eigenvector values of a principal nization but not cell transdifferentiation. In contrast, CTCF component analysis (PCA) on the Hi-C correlation matrix (‘PC1 depletion in induced macrophages impairs the full-blown values’). Overall, although most of the genome remained stable, upregulation of inflammatory genes after exposure to endo- around 14% of A or B compartments were dynamic during transdif- toxin. Our results demonstrate that CTCF-dependent genome ferentiation, showing transcriptional changes correlating with the topology is not strictly required for a functional cell-fate con- altered compartmentalization (Fig. 1b–f, Extended Data Fig. 1a–d version but facilitates a rapid and efficient response to an and Supplementary Note 2). Next, we used chromosome-wide external stimulus. insulation potential26 to identify between 3,100 and 3,300 TAD bor- Lineage-instructive transcription factors establish new cell iden- ders per time point (Fig. 1g). Boundaries were highly reproducible tities by activating a new gene expression program while silencing between biological replicates (Jaccard index > 0.99) and enriched the old one. Whereas this is largely achieved through binding to in binding sites for CTCF (Extended Data Fig. 1e). Genome-wide promoters and enhancers, genome topology has recently emerged insulation scores analyzed by PCA over time revealed progressive as a new player in gene regulation. Chromatin contact maps changes, reflecting a transdifferentiation trajectory (Extended Data obtained by chromosome conformation capture techniques, such Fig. 1f). While 70% of TAD borders were stable across all stages, as Hi-C, revealed that chromatin can be separated at the megabase 18% were lost or gained and 12% were transiently altered (Fig. 1g). (Mb) level into active (A) and inactive (B) compartments19, which CTCF binding was substantially more enriched at stable than at are themselves subdivided into TADs. Large deletions overlapping dynamic boundaries (Fig. 1h), as observed earlier27. Furthermore, boundaries can cause fusion of adjacent domains, which can lead to while lost borders showed some CTCF occupancy in B cells that developmental abnormalities20. In addition, inversion or deletion of decreased in iMacs, gained borders were depleted for CTCF in both individual CTCF-binding sites can induce a loss of specific contacts cell states (Fig. 1h), indicating CTCF-independent mechanisms or insulation from active chromatin21–23. Mechanistically, genomic driving local insulation. The dynamic rearrangement of TAD bor- insulation by TADs is thought to facilitate enhancer–promoter inter- ders during transdifferentiation is illustrated by the DDX54 locus actions while inhibiting cross-boundary communication between (Fig. 1i), in which a new boundary appears in iMacs without appar- regulatory elements to prevent aberrant gene activation1. Hence, ent changes in CTCF binding. Furthermore, border gain or loss the importance of CTCF and TAD organization in facilitating did not correlate with changes in local gene expression (Extended 1Centre for Genomic Regulation (CRG) and Institute of Science and Technology (BIST), Barcelona, Spain. 2Josep Carreras Leukaemia Research Institute (IJC), Barcelona, Spain. 3CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain. 4Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain. 5Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. 6ICREA, Barcelona, Spain. 7Department of Pulmonary Medicine, Erasmus MC, Rotterdam, the Netherlands. 8Department of Cell Biology, Erasmus MC, Rotterdam, the Netherlands. 9Universitat Pompeu Fabra (UPF), Barcelona, Spain. ✉e-mail: [email protected]; [email protected]; [email protected] NatURE GENETICS | www.nature.com/naturegenetics LETTERS NATURE GENETICS a bc Chr6:100–150 Mb +β-est iMac 0 h CEBPA 168 h 0 B cell 10–4 10–5 10–6 0 iMac A B B A B cell iMac B cell de f B AAA B B A B A B n = 980 n = 1,815 n = 702 n = 1,102 B cell –16 P < 2.2 × 10 –12 1 100% P = 1.6 × 10 –13 9 h 2.5 P = 2 × 10 18 h B A ratio) 24 h 2 0 48 h 72 h Stable Dyn A B 0 96 h 86% 14% PC2 16.4% 120 h –1 144 h A B A −2.5 B cell iMac B A B iMac 0% RNA (centered log –1 0 1 iMac iMac iMac iMac PC1 45.0% B cell B cell B cell B cell i gh ∆Hi-C signal (iMac – B cell) 40 4,000 3 Stable 30 0 3,000 (n = 2,509) Stable Transient –3 Transient 20 (n = 391) Contact differential 2,000 Gained Gained Lost (n = 581) 10 112 Mb HECTD4 PTPN11 OAS1 DDX54 TPCN1 SDS RBM19 114 Mb 1,000 CTCF coverage chr 12 (per kb of border) Lost (n = 525) B cell 0 0 Percentage of TAD borders B cell iMac B cell iMac CTCF iMac Fig. 1 | Transcription-factor-driven transdifferentiation rewires nuclear compartments and modulates TAD borders independently of CTCF binding. a, Schematic overview of the transdifferentiation system. CEBPA-ER in B cells (BLaER cell line) translocates to the nucleus after β-estradiol (β-est) treatment, activating the factor. A week after treatment the cells convert into induced macrophages (‘iMac’ stage). b, Representative in situ Hi-C contact maps (100-kb resolution) of a 50-Mb DNA region of B cells and iMacs. The color scale represents the normalized number of contacts per read. c, Transformation of the Hi-C map on the basis of the PC1 values of a PCA on the Hi-C correlation matrix. PC1 values for A and B compartments are shown in yellow and blue, respectively; dotted rectangles highlight local compartment changes during transdifferentiation. d, PCA of PC1 compartment values (n = 28,749 bins), with the beige arrow indicating the transdifferentiation trajectory. e, Proportion of dynamic compartment bins (Dyn), including the distribution of its different subcategories. f, Integration of gene expression associated with the dynamic compartment using RNA-seq (n represents the number of genes and P values are calculated using a two-sided Wilcoxon rank-sum test). g, Number of stable, transiently changed, gained or lost TAD borders in B cells and iMacs. h, CTCF-peak coverage at the different types of borders (n represents the number of borders in each category) in B cells and iMacs. i, Differential contact map (iMac minus B cell signal) at the DDX54 locus (top). Color scale represents differential contacts per 100,000 reads. Snapshot of genome browser showing CTCF ChIP–seq signals at the locus (bottom). CTCF peaks at the newly created border are highlighted. All box plots depict the first and third quartiles as the lower and upper bounds of the box, with a thicker band inside the box showing the median value and whiskers representing 1.5× the interquartile range. Data Fig. 1g), indicating that transcription is not a driver of the sulfoxide (DMSO) (as a control) at 24 and 168 h postinduction observed changes. However, whereas motif analysis at ATAC-seq (hpi) of transdifferentiation. Scaling of contact probabilities as a peaks within stable borders did indeed show a strong enrichment function of genomic separation did not change after CTCF deple- for the CTCF motif, dynamic borders were enriched for PU.1 and tion (Extended Data Fig. 2c). Analysis of chromosome-wide insu- EBF1 motifs (Extended Data Fig. 1h), raising the possibility that lation potential in wild-type and CTCF-AID B cells showed that lineage-restricted transcription factors are involved in disrupting fusing the mAID tag to CTCF only had a negligible impact on and/or establishing these borders. TAD organization (Extended Data Fig. 2d,e). However, ~70% of To directly assess the importance of CTCF during TAD borders became undetectable and not visible in Hi-C con- CEBPA-induced transdifferentiation we devised an auxin-inducible tact maps after auxin treatment, both at 24 hpi and at iMac stages degron approach28 (Fig.